Dependence on the use of zygotic seed in breeding programs, where success relies on seed availability, particularly that of genetically superior seeds, often leads to low returns on investment. The situation is exacerbated in tree breeding and improvement programs especially where conifer species are used, because the time span from flower bud initiation to seed maturation is usually one to three years. Climatic events and pest infection contribute to seed production variability from year to year (Harlow and Harrar 1968).
The development and advancement of somatic embryogenesis as a vegetative propagation technology has made it possible to mass-produce genetically identical individuals through the asexual reproduction of a source explant (Tautorus et al. 1991, Roberts et al. 1995). This technology allows for the application of clonal forestry in plantation programs. The primary advantages of clonal forestry as defined by Kleinschmit et al. (1993) and Park et. al. (1998a) are: 1) the ability to capture a greater portion of the non-additive genetic gain from selected individuals within a breeding population; 2) the capability to rapidly introduce individuals with desirable traits to meet known site conditions; and 3) the ability to carefully plan genetic diversity into plantation programs. The primary challenge in utilising somatic embryogenesis for clonal forestry in plantation programs is the development of cost effective and scaleable methods of somatic embryo culture to produce autotrophic and acclimatised seedlings.
Somatic embryogenesis of woody plants is generally a multi-step process (reviewed by Sutton and Polonenko, 1999; U.S. Pat. Nos. 4,957,866; 5,183,757; 5,294,549; 5,413,930; 5,464,769; 5,482,857; 5,506,136; the disclosure of all of which are herein incorporated by reference). No matter how diverse the different somatic embryogenesis protocols might be, the one common step is that somatic embryos must be germinated to produce somatic seedlings.
For zygotic embryos in natural seed, germination is supported by stored nutrients within the endosperm (in angiosperms) or megagametophyte (in gymnosperms). Major forms of storage nutrients in the nutritive tissues are starch, proteins and lipids which are broken down into simple substrates for us in various biochemical and physiological activities during germination. For somatic embryos, nutrients needed to support germination must be supplied by a nutrient medium during somatic embryo culture. There are two standard approaches for germinating somatic embryos. The first approach utilises conventional in vitro methods and is generally comprised of the following steps. First, a naked somatic embryo (i.e., an embryo unprotected by any coatings) is sown, using aseptic techniques, onto sterilised semi-solid or liquid media contained within a solid-support such as a Petri dish or a phytatray under sterile conditions. Second, after the somatic embryo has germinated and grown under sterile conditions, the young seedling is transplanted into conventional nursery growing systems. The second approach utilises encapsulation (generally gel-encapsulation) of the somatic embryos (Carlson and Hartle 1995, Gray et al., 1995; U.S. Pat. Nos. 4,562,663; 4,777,762; 4,957,866; 5,010,685; 5,183,757; 5,236,469; 5,427,593; 5,451,241; 5,486,218; 5,482,857 all of which are herein incorporated by reference) prior to germination. The embryos are encapsulated in various coating materials to form so-called “artificial seed”, “synthetic seed” or “manufactured seed”. This encapsulation process may or may not incorporate nutrients into the encapsulating medium, and provides a means by which the embryos can presumably be sown with conventional nursery seeding equipment (i.e., drum seeders or fluid drill seeders) into conventional nursery growing systems. The prior art makes references to sowing artificial seeds ex vitro into germination media comprised of soil or soil-less mixes, but in fact, the prior art only teaches methods for germinating artificial seeds in vitro, i.e., on sterilised semi-solid laboratory media. No approaches are taught or otherwise disclosed in the prior art for sowing encapsulated somatic embryos and/or artificial seed and/or manufactured seed into conventional growing systems using conventional sowing equipment.
The past dependence of somatic embryo germination on in vitro methods stems from the anatomical distinction between somatic embryos and zygotic seeds: a somatic embryo lacks the nutritive tissues and the protective seed coat that a zygotic seed possesses. Consequently, somatic embryos had to rely on exogenous nutrient supply for germination and early growth and these events had to take place in sterile environments in vitro for protection against both physical and biological damaging agents such as environmental stresses and microbial pathogens.
There are many disadvantages associated with in vitro protocols. The most significant are: 1) the repeated manual handling of each individual embryo in the germination and transplanting steps; 2) the stringent requirement for sterile techniques and culture conditions through all steps until somatic germinants are transplanted out of the in vitro germination environment into horticultural growing media; and 3) the difficulty in acclimatizing in vitro plantlets into ex vitro nursery environments. Therefore, the art of traditional in vitro protocols has an inherent nature of low efficiency and high cost, characteristics that are prohibitive to mass production of somatic seedlings. These undesirable characteristics make the commercial production of somatic seedlings less competitive than that of the zygotic seedlings (Sutton and Polonenko 1999). Automation, including robotics and machine vision, may reduce or eliminate the extensive manual-handling that is currently necessary to germinate naked somatic embryos. However, no commercial equipment currently exists which can reliably, aseptically, and cost-effectively perform the in vitro protocols for germination and gorwht of naked somatic embryos and subsequent transplanting of seedlings into conventional propagation systems (Roberts et al., 1995; reviews by Sakamoto et al., 1995; and Sutton and Polonenko 1999).
There are also numerous biological and operational disadvantages inherent in using gel-encapsulated somatic embryos. Biologically, the most significant disadvantage is the much lower germination vigour and conversion success into plants than corresponding zygotic seeds, as seen in the prior art protocols for encapsulating or otherwise coating somatic embryos (Redenbaugh et al., 1993; Carlson & Hartle, 1995; Gray et al., 1995). This is in sharp contrast with the germination vigour and conversion success of non-encapsulated or non-coated somatic embryos, produced with methods disclosed in the art, and then sown using aseptic techniques onto in vitro germination media in sterile conditions. The conversion rates of germinants from in vitro sown somatic embryos can approximate those of the corresponding zygotic seeds (e.g., greater than 85%) (Gupta and Grob, 1995).
Timmins et al. (U.S. Pat. No. 5,119,588, incorporated herein by reference) recognised that “somatic embryos are too under-developed to survive in a natural soil environment” and therefore must be “cultured with an energy source, such as sucrose”. They identify a method by which plant somatic embryos can be sown into horticultural containers filled with particulate soil-like substrates. Solutions containing compounds serving as carbon and energy sources and other nutrients, such as minerals and vitamins, are added to the substrates before or after the embryos are sown. Because such a “culture medium is highly susceptible to invasion by phytopathogens, which can result in death or retard the growth of the embryos”, they teach that the containers, substrate, nutrient solutions and other components of their system must be biologically sterile. Somatic embryos must be sown into containers using aseptic techniques. Each sown container must be kept biologically separated from the others and from the external environment and must be kept in a sterile condition until the embryo has successfully germinated and developed into a complete, independent autotrophic plant. Only after autotrophy has been reached can the somatic seedlings be removed from the sterile conditions and then transplanted into a conventional commercial propagation environment. Even though the art taught by such methods may be practised to produce somatic seedlings, such methods are labour-intensive and bear characteristics of low efficiency, high cost and impracticability for mass production of somatic seedlings in a nursery environment.
The inventors named herein have previously discovered that plant somatic embryos can be directly sown ex vitro in a variety of soil or soil-like growing media in non-sterile conditions (PCT patent application no. WO 09/965293A1, incorporated herein by reference), particularly plant somatic embryos that have been pre-germinated (U.S. Pat. No. 6,444,467 and U.S. patent application Ser. No. 09/550,110). In these patents and patent applications, exogenous energy and nutrient sources are still delivered to plant somatic embryos after they have been directly sown ex vitro in soil or soil-like growing media to facilitate and maximize the ex vitro growth of, somatic embryos.
Nevertheless, there is a constant need for improvement of these techniques and methods in order to overcome the disadvantages of the germination and growth phases associated with somatic plant embryos.